Membrane-associated immunogens of mycobacteria

ABSTRACT

Nucleic acid encoding four novel immunodeterminant protein antigens of  M. bovis  BCG, which is a vaccine strain for tuberculosis, have been isolated. These genes were isolated as immunoreactive recombinant clones from a genomic library of  M. bovis  BCG DNA, constructed in pBR322 vector, and screened with sera collected from tuberculosis patients. The BCG DNA insert of one of the recombinants, pMBB51A, which expressed an antigen of Mr 90 kD, was sequenced completely and an ORF encoding 761 amino acids encoding a protein of deduced molecular weight 79 kD, was identified. This gene was identified to encode a membrane bound, ion-motive ATPase of  M. bovis  BCG. The approach described here can be used to identify immunogens of mycobacteria. In addition, the well-characterized  M. bovis  BCG antigens can be used in the prevention, diagnosis and treatment of tuberculosis. The 79 kD antigen is also useful in the design of recombinant vaccines against different pathogens. The sequence of the 79 kD membrane-associated polypeptides also are useful for the development of specific PCR amplification based diagnostic procedures for the detection of mycobacteria. Also, the promoter of the 79 kD antigen is useful for expressing homologous and/or heterologous antigens in mycobacteria.

TECHNICAL FIELD OF THE INVENTION

[0001] The invention relates to membrane-associated polypeptides ofmycobacteria and, in particular, the use of such polypeptides and thenucleic acids encoding them for use as vaccines and diagnostic reagents.

BACKGROUND OF THE INVENTION

[0002] The mycobacteria are a diverse collection of acid fast,gram-positive bacteria, some of which cause important human and animaldiseases. In humans, the two most common mycobacteria-caused diseasesare tuberculosis (TB) and leprosy, which result from infection with M.tuberculosis and M. leprae, respectively.

[0003] Tuberculosis displays all of the principal characteristics of aglobal epidemic disease. Currently, tuberculosis afflicts more than 35million individuals worldwide and results in over 4 million deathsannually. In India, at any given time, almost 8 million people arereported to suffer from this disease and 500,000 deaths recorded. Thesefigures may not cover the totality of those suffering from this diseasein this country. Thus, tuberculosis appears to be a problem of majorconcern in India as also in many other countries of the world.

[0004] Tuberculosis is caused by M. tuberculosis, M. bovis, M. africanumand M. microti, the acid-fast, Gram positive, tubercle bacilli of thefamily Mycobacteriaceae. Some local pathogenic strains of M.tuberculosis have also been isolated from patients in Madras and othercities in India, which differ in some respects from M. tuberculosisH37Rv, which is a virulent strain.

[0005] In recent years, certain groups of individuals with AIDS havebeen found to have a markedly increased incidence of TB as well. It hasnow been shown that one group of mycobacteria which consists of M.avium, M. intracellulare and M. scrofulaceum, jointly known as MAIScomplex, is responsible for disseminated disease in a large number ofpersons with AIDS (Kiehn et al., J. Clin. Microbiol., 21:168-173 (1985);Wong et al., Amer. J. Med., 78:35-40 (1985)).

[0006] Since Koch identified M. tuberculosis as the causative agent oftuberculosis in 1882, many scientific studies and public health effortshave been directed at diagnosis, treatment and control of this disease.However, characteristics of M. tuberculosis have hampered research toimprove diagnosis and to develop more effective vaccines. In addition,the biochemical composition of the organism has made identification andpurification of the cellular constituents difficult, and many of thesematerials once purified, lack sensitivity and specificity as diagnosticreagents. As a result, diagnostic and immunoprophylactic measures formycobacterial diseases have changed little in the past half century. Theconventional methods for the diagnosis of M. tuberculosis aretroublesome and results are delayed.

[0007] Bacillus Calmette-Guerin (BCG), an avirulent strain of M. bovis(Calmette, A., Masson et Cie, Paris (1936)), is used extensively as avaccine against tuberculosis. Though numerous studies have found that ithas protective efficacy against tuberculosis (Luelmo, F., Am. Rev.Respir. Dis., 125, 70-72 (1982)) BCG has failed to protect againsttuberculosis in several trials (WHO, Tech. Rep. Ser., 651:1-15 (1980))for reasons that are not entirely clear (Fine, P., Tubercle, 65:137-153(1984); Fine, et al., Lancet, (ii):499-502 (1986)).

[0008] The eradication with vaccination, early diagnosis, and efficienttherapy is an important objective of the drive to combatmycobacterioses. The lacunae in the present knowledge of the biology ofthese pathogens—their make-up, their natural history, their physiology,biochemistry and immunological reactivities, highlights the need forattempts to unravel their weaknesses, so that more efficient ways tocombat this disease can be devised. To develop more effective tools forthe diagnosis and prevention of these diseases, it is important tounderstand the immune response to infection by mycobacterial pathogens.The mycobacterial components that are important in eliciting thecellular immune response are not yet well defined. The antibody andT-cell responses to infection or inoculation with killed mycobacteriahave been studied in humans and in animals. Human patients with TB orleprosy produce serum antibodies directed against mycobacterialantigens. Although antibodies may have some function in theantimycobacterial immune response, the exact function remains to beclarified since no protective role can be ascribed to these antibodies.Protection against mycobacterial diseases involves cell-mediatedimmunity.

[0009] Mycobacteria do not produce any directly toxic substances andconsequently their pathogenicity results from multiple factors involvedin their interaction with the infected host. Intracellular parasitismprobably depends on host cell trophic factors; it is conceivable thattheir short supply may be bacteriostatic and could play a role in themechanism of mycobacterial dormancy.

[0010] It is generally understood that protective immunity inmycobacterial infection is mediated by specific T cells which activatemacrophages into non-specific tuberculocidal activity. Evidence suggeststhat gamma-INF triggers macrophages towards H₂O₂-mediated bacterialkilling, but related or other macrophage activating factor (MAF)molecules may also be involved. The causes responsible for theinadequate bactericidal function at sites of abundant T cellproliferation have not yet been explained. Dissociation betweendelayed-type hypersensitivity (DTH) and protective immunity led to viewsthat T-cells of a distinct subset or specificity could be responsiblefor the acquired resistance to mycobacterial infection. Alternatively,interference with protection may result from corollary cellularreactions, namely by suppressor T-cells and macrophages, or from theshifting of T-cells towards helper function for B-cells.

[0011] Unlike viral and some parasite pathogens which can evade hostresistance by antigenic shift, mycobacteria have a resilient cell wallstructure and can suppress host immune responses by the action of theirimmunomodulatory cell wall constituents. Whilst the success ofprotective immunization towards other microbial pathogens mainly dependson quantitative parameters of immunity, it appears that mycobacterialimmunomodulatory stimuli produce a regulatory dysfunction of the hostimmune system. This may not be possible to override simply by moreresolute immunization using vaccines of complex composition such aswhole mycobacteria (e.g. BCG). Perhaps mycobacteria did not evolvepotent “adjuvant” structures to boost the host immunity but rather tosubvert host defenses towards ineffective cellular reactions operatingto the advantage of the pathogen. Vaccination with an attenuatedpathogen such as BCG could amplify further immune responses but withlimited protection of the host, the potential scope for immunizationwith defined antigens is yet to be explored.

[0012] The purification and characterization of individual antigenicproteins are essential in understanding the fundamental mechanism of theDTH reaction on the molecular level. The possible functional role ofproteins of defined structure in the pathogenesis of mycobacterialdiseases as well as for diagnostic purposes remains of great interest.Numerous groups have attempted to define mycobacterial antigens bystandard biochemical and immunological techniques, and common as well asspecies specific antigens have been reported in mycobacteria (Minden, etal., Infect. Immun., 46:519-525 (1984); Closs, et al., Scand. J.Immunol., 12:249-263 (1980); Chaparas, et al., Am. Rev. Respir. Dis.,122:533 (1980); Daniel, et al., Microbiol. Rev., 42:84-113 (1978);Stanford, et al., Tubercle, 55:143-152 (1974); Kuwabara, S., J. Biol.Chem., 250:2556-2562 (1975)).

[0013] Very little information about the mycobacterial genome isavailable. Initially, basic studies were conducted to estimate thegenome size, G+C content and the degree of DNA homology between thevarious mycobacterial genomes (Grosskinsky, et al., Infect. Immun., 57,5:1535-1541 (1989); Garcia, et al., J. Gen. Microbiol., 132:2265-2269(1986); Imaeda, T., Int. J. Sys. Bacteriol., 35, 2:147-150 (1985);Clark-Curtiss, et al., J. Bacteriol., 161 3:1093-1102 (1985); Baess, I.et al., B., Acta. Path. Microbiol. Scand., (1978) 86:309-312; Bradley,S. G., Am. Rev. Respir. Dis., 106:122-124 (1972)). Recently, recombinantDNA techniques have been used for the cloning and expression ofmycobacterial genes. Genomic DNA fragments of M. tuberculosis, M. lepraeand some other, mycobacterial species were used for the construction oflambda gtll phage (Young, et al., Proc. Natl. Acad. Sci., U.S.A.,82:2583-2587 (1985); Young, et al., Nature (London), 316:450-452 (1985))or other vector-based recombinant gene libraries. These libraries werescreened with murine monoclonal antibodies (Engers, et al., Infect.Immun., 48:603-605 (1985); Engers, et al., Infect. Immun., 51:718-720(1986)) as well as polyclonal antisera and some immunodominant antigenswere identified. The principal antigen among these being five 12, 14,19, 65 & 71 kDa of M. tuberculosis (Young et al., Proc. Natl. Acad.Sci., U.S.A., 82:2583-2587 (1985); Shinnick et al., Infect. Immun.,55(7):1718-1721 (1987); Husson and Young, Proc. Natl. Sc. Acad.,84:1679-1683 (1987); and five 12, 18, 23, 36 & 65 kDa antigens of M.leprae (Young, et al., Nature (London), 316:450-452 (1985)). A fewhomologues of some of these antigens were also identified in some othermycobacterial species (e.g., BCG) (Yamaguchi et al., FEB 06511,240:115-117 (1988); Yamaguchi et al., Infect. Immun., 57:283-288 (1989);Matsuo, et al., J. Bacteriol., 170, 9:3847-3854 (1988); Radford, et al.,Infect. Immun., 56, 4:921-925 (1988); Lu, et al., Infect. Immun., 55,10:2378-2382 (1987); Minden, et al., Infect. Immun., 53, 3:560-564(1986); Harboe, et al., Infect. Immun., 52, 1:293-302 (1986); Thole, etal., Infect. Immun., 50, 3:800-806 (1985)). These antigens, however, areeither intracellular or secreted molecules.

[0014] Although M. bovis BCG has been widely used as a vaccine againsttuberculosis, the determination of the membrane-associated polypeptidesof mycobacterium that are capable of inducing a protective immuneresponse is highly desirable. The use of such a membrane-associatedpolypeptide or the DNA encoding it provides for the generation ofrecombinant vaccines, e.g., mycobacterial membrane-associated immunogensexpressed in, for example, a virus or bacterium such as vaccinia virus,Salmonella, etc. used as a live carrier, or the display ofnon-mycobacterial immunogens on the surface of a cultivablemycobacterial strain which can be used as a live recombinant vaccine.

[0015] Accordingly, it is an object herein to provide methods foridentifying and isolating nucleic acids encoding a membrane-associatedpolypeptide of mycobacteria.

[0016] Further, it is an object herein to provide membrane-associatedpolypeptides of mycobacteria and the nucleic acids encoding it.

[0017] Still further, it is an object herein to provide vaccinesutilizing all or part of the membrane-associated polypeptide of amycobacterium or the DNA encoding such membrane-associated polypeptide.

[0018] Still further, it is an object to provide reagents comprisingsaid membrane-associated polypeptide with a mycobacterium or DNAencoding it useful in diagnostic assays for mycobacterial infection.

[0019] Still further, it is an object to provide a promoter sequencecomprising the promoter of said membrane associated polypeptide, whichcan direct gene expression in mycobacteria as well as in othermicroorganisms such as E. coli.

SUMMARY OF THE INVENTION

[0020] In accordance with the foregoing objects, the invention includescompositions comprising nucleic acid encoding all or part of amembrane-associated polypeptide of a mycobacterium and themembrane-associated polypeptide encoded by said DNA. Themembrane-associated polypeptide is characterized by the ability todetect an immune response to pathogenic mycobacteria or the mycobacteriafrom which the membrane associated polypeptide or part thereof isderived. Such mycobacteria include M. bovis, M. tuberculosis, M. leprae,M. africanum and M. microti, M. avium, M. intracellular and M.scrofulaceum and M. bovis BCG.

[0021] A particular mycobacterial membrane-associated polypeptide is a79 kD ion-motive ATPase. Extra-cellular, intra-cellular andtransmembrane domains are identified in this mycobacterialmembrane-associated polypeptide based upon its DNA and deduced aminoacid sequence.

[0022] The invention also includes vaccines utilizing all or part of amembrane-associated mycobacterial polypeptide or an expressible form ofa nucleic acid encoding it. The invention also includes mycrobacterialpromoter sequences capable of directing gene expression in mycobacteriaas well as in other microorganisms such as E. coli. Such promoters arefrom mycobacterial genes encoding membrane-associated ATPases. Apreferred promoter is that of the gene encoding the M. bovis BCG 79 kDmembrane-associated polypeptide. This promoter sequence is especiallyuseful to express genes of interest in mycobacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 illustrates the results of immunoscreening of recombinantcolonies carrying M. bovis BCG DNA (panel A) and M. tuberculosis H37RvDNA (panel B), using sera from TB patients in which the presence of M.bovis BCG antigens and M. tuberculosis H37Rv antigens capable ofreacting with the antisera is indicated by a qualitative signal.

[0024]FIG. 2 shows the comparison of restriction site maps ofrecombinant clones carrying BCG DNA identified using the immunoscreeningassay described herein (panel B) with the restriction site maps of fiveimmunodominant antigens of M. tuberculosis and M. bovis BCG genomicDNAs, respectively, (Husson and Young, Proc. Natl. Acad. Sci., U.S.A.,84:1679-1683 (1987); Shinnick et al., Infect. Immun., 55:1718-1721(1987) (panel A)). Restriction maps in each panel have been drawn to thesame scale (indicated at the top), and restriction sites are indicatedabove the restriction maps. The dotted line in panel A represents thenon-mycobacterial DNA. Restriction enzymes: B, BamHI, E, EcoRI, G,BglII, K, KpnI, P, PvuI, X, XhoI, H, HincII, U, PvuII, Ps, PstI, Hi,HindIII. In panel A, A is SalI and S is SacI. In panel B, S is SalI.

[0025]FIG. 3 illustrates the results of Western blot analysis of thesonicated supernate of recombinant clone pMBB51A which carries a BCG DNAinsert identified following immunoscreening of the recombinant colonies.The top panel shows reactivity of MBB51A (lane 2) and E. coli (lane 1)with sera from TB patients. The bottom panel (part A) shows reactivityof MBB51A (lanes 1 and 2) and E. coli (lane 3) with anti-H37Rv seraraised in rabbits. Part B shows reactivity of MBB51A (lanes 1 and 2) andE. coli (lane 3) with the second antibody alone. Arrows indicate theposition of the 90 kD immunoreactive BCG protein expressed by therecombinant MBB51A, which was absent in the negative control.

[0026]FIG. 4 illustrates the nucleotide sequence (Seq. ID No.: 1) ofclone pMBB51A 3.25 kb insert DNA containing the M. bovis BCGimmunoreactive MBB51A gene encoding an ion-motive ATPase, with a deducedmolecular weight of 79 kD. The deduced amino acid sequence (Seq. ID No.:2) is shown below the nucleotide sequence. Upstream promoter elementsare underlined. Transcription termination region is indicated byinverted arrows. 5′ and 3′ flanking regions are also shown.

[0027]FIG. 5 illustrates a schematic model derived for the 79 kD proteinencoded by pMBB51A which represents an ion-motive ATPase of BCG. Themodel considers only the structural and functional features that areprominent in the other ion-motive ATPase homologs of transmembranedomains of the protein. Functionally, important amino acid residues areindicated (P), proline at position 400; (D), aspartic acid at position443; (G), glycine at position 521; and (A), alanine at position 646.Numbers indicate amino acid residues broadly defining the limits of thetransmembrane domains.

[0028]FIG. 6 illustrates the results of Southern blot hybridization ofBamHI digest of genomic DNAs from M. bovis BCG (lane 6), M. tuberculosisH37Rv (lane 5), M. smegmatis (lane 4) and M. vaccae (lane 3 usingpMMB51A DNA insert (lane 8) as probe. Panel A shows ethidium bromidestained gel and panel B shows the results of Southern blothybridization.

DETAILED DESCRIPTION OF THE INVENTION

[0029] As used herein, a “membrane-associated polypeptide” of amycobacterium is defined as any Mycobacterial membrane-associatedpolypeptide which is capable of detecting an immune response against thewild-type mycobacterium containing the membrane-associated polypeptide.However, based upon the observed cross-reactivity of the 79 kDmembrane-associated polypeptide of an M. bovis BCG with pooled anti-serafrom patients afflicted with tuberculosis and the cross-hybridization asbetween the DNA encoding the 79 kD membrane-associated polypeptide andthe DNA of M. tuberculosis H37Rv, the membrane-associated polypeptide ofthe invention is not limited to that identified herein from M. bovisBCG. Rather, it encompasses not only homologs to the 79 kD ion-motiveATPase but also any and all membrane-associated polypeptides of amycobacterium that can be used to detect an immune response by the sameor a different mycobacteria in which the membrane-associated polypeptideis normally found.

[0030] As used herein, “nucleic acid” includes DNA or RNA as well asmodified nucleic acid wherein a detectable label has been incorporatedor wherein various modifications have been made to enhance stability,e.g., incorporation of phosphorothioate linkages in the phosphoribosebackbone, etc. Such nucleic acid also includes sequences encoding theanti-sense sequence of the DNA encoding the membrane-associatedpolypeptide such that the now well-known anti-sense technology can beused to modulate expression of such membrane-associated polypeptides.

[0031] In some aspects of the invention, the nucleic acid sequenceencoding all or part of a membrane-associated polypeptide of themycobacterium is used as a vaccine.

[0032] When so-used the nucleic acid is generally an “expressiblenucleic acid” that contains all necessary expression regulationsequences to control transcription and translation of the nucleic acidin a designated host system. In some vaccine embodiments, the DNAencodes a chimeric polypeptide containing at least one transmembranedomain of the membrane-associated polypeptide and an “immunogenicpolypeptide”. The transmembrane domain is used to display theimmunogenic polypeptide on the surface of a particular host organismsuch as an attenuated live vaccine. When the membrane-associatedpolypeptide includes more than one transmembrane region, one or more ofthe transmembrane regions can be used with an immunogenic polypeptide.Thus, for example, the 79 kD ion-motive ATPase as shown in FIG. 5 has atleast three extracellular domains into which an immunogenic polypeptidecan be engineered by well-known methods involving recombinant DNAtechnology. Although it is preferred that more than one transmembraneregion be used to display an immunogenic polypeptide, one skilled in theart can readily vary the length of such a membrane-associatedpolypeptide to maximize an immunogenic response or to minimize theamount of membrane-associated polypeptide used in such applications.

[0033] As used herein, “immunogenic polypeptide” comprises all or partof any polypeptide which can potentially be utilized in a vaccine ordiagnostic application. Thus, the immunogenic polypeptide can compriseheterologous immunogens, i.e., immunogens from non-mycobacterialsources, e.g., Salmonella or Shigella or from different mycobacteriafrom which the membrane-associated polypeptide is derived, e.g.,immunogens from Mycobacterium tuberculosis fused to amembrane-associated polypeptide from M. bovis BCG. However, in someinstances homologous immunogens can be used. For example, each of theextracellular domains as set forth in FIG. 5 herein can be combined anddisplayed by combination with one or more of the transmembrane domainsfrom the membrane-associated polypeptide normally containing them.Alternatively, the intercellular domains can be displayedextracellularly using appropriate transmembrane regions from the samemolecule.

[0034] In an alternate vaccine embodiment, all or part of themembrane-associated polypeptide of mcobacteria, rather than the DNAencoding, is used as part of a vaccine. Such proteinaceous vaccines areformulated with well-known adjuvants and administered followingwell-established protocols known to those skilled in the art.

[0035] In still other embodiments, the nucleic acid encoding themembrane-associated polypeptide of the invention can be used as adiagnostic for detecting infection based upon hybridization withwild-type genes contained by the infectious mycobacterium. Suchdetection can comprise direct hybridization of DNA extracted from anappropriate diagnostic sample or PCR amplification using the nucleotidesequence of the nucleic acid encoding the membrane-associatedpolypeptide of the invention to prime amplification. If PCRamplification is primed in a conserved region the presence ofmycobacteria in a diagnostic sample can be determined. If primed in anon-conserved region which is species specific the diagnostic assaydetermined the specific mycobacterium causing an infection.

[0036] In addition, the membrane-associated polypeptide of the inventioncan also be used to detect the presence of antibodies in the sera ofpatients potentially infected with mycobacteria. Such detection systemsinclude radioimmunoassays and various modifications thereof which arewell-know to those skilled in the art. In addition, themembrane-associated polypeptide of the invention can be used to detectthe presence of a cell-mediated immune response in a biological sample.Such assay systems are also well-known to those skilled in the art andgenerally involve the clonal expansion of a sub-population of T cellsresponding to stimuli from the membrane-associated polypeptide. Whenso-used, the humoral and/or cell-mediated response of a patient can bedetermined and monitored over the course of the disease.

[0037] Recombinant clones encoding immunogenic protein antigens of M.bovis BCG have been isolated from a genomic library of M. bovis BCG DNA.In particular, DNA fragments encoding four protein antigens of M. bovisBCG have been isolated by probing a pBR322 library of M. bovis BCG DNAwith sera from TB patients, absorbed on E. coli. Restriction site mapsof these four recombinant clones are different from those of the fiveimmunodominant antigens of mycobacteria (Young, et al., Proc. Natl.Acad. Sci., U.S.A., 82:2583-2587 (1987); Husson and Young, Proc. Natl.Acad. Sci., U.S.A., 84:1679-1683 (1987); Shinnick et al., Infect.Immun., 55:1718-1721 (1987)), thereby indicating that these clonedprotein antigens are novel. One of the recombinant DNA clones encoded animmunoreactive protein with apparent molecular weight of 90 kD asdetermined by Western blot analysis. The complete nucleotide sequence ofthe insert DNA of this clone was determined. This clone was found tocarry a mycobacterial promoter and a monocistronic ORF encoding aprotein of 761 amino acids with a deduced molecular weight of 79 kD.This 79 kD protein had extensive homology with ion-motive ATPases of S.faecalis (Solioz et al., J. Biol. chem, 262:7358-7362 (1987)), E. coli(Hesse et al., Proc. Natl. Acad. Sci., U.S.A., 81:4746-4750 (1984)) andseveral other organisms, and thus, represents an ion-motive ATPase or aputative K+ATPase of BCG. Using computer algorithms, this ion-motiveATPase was determined to be a membrane protein and has a homologue in M.tuberculosis H37Rv, which is pathogenic in humans, but not in M. vaccaeand M. smegmatis, which are non-pathogenic. As a result, novel BCGimmunogens can be available which can be useful in the prevention,diagnosis and treatment of tuberculosis and other mycobacterialinfections. They can be used, for example, in the development of highlyspecific serological tests for screening patients for individualsproducing antibodies to M. tuberculosis, or those infected with M.tuberculosis, in the development of vaccines against the disease, and inthe assessment of the efficacy of the treatment of infected individuals.

[0038] Further, based on the nucleotide sequence of the pMBB51A insertDNA, appropriate oligonucleotide primers can be used for PCRamplification using as template M. bovis BCG or M. tuberculosis H37RvDNA. Such a PCR amplification scheme can be thus useful for thedetection of mycobacterial DNA in a given sample. Further, by ajudicious choice of the primer design, such an amplification procedurecan be adapted for taxonomic classification of mycobacterial DNAs. Forexample, using primers to flank a heavily conserved region such as theATP-binding site, PCR amplification is common to all mycobacterialspecies, whereas using primers from non-conserved areas, amplificationcan be made species specific.

EXAMPLE I Isolation and Characterization of Genes Encoding ImmogenicProtein Antigens of Mycobacterium bovis BCG and Mycobacteriumtuberculosis H37R

[0039] A. Construction of Recombinant DNA Libraries of M. bovis BCG DNAand Mycobacterium tuberculosis H37Rv

[0040] A recombinant DNA library of M. bovis BCG genomic DNA wasconstructed using pBR322 a high copy number plasmid vector (Bolivar, etal., Gene, 2:95-113 (1977)) with antibiotic markers (ampicillin andtetracycline) and several unique cloning sites. M. bovis BCG cells wereharvested from a culture in late logarithmic phase of growth and highmolecular weight DNA was isolated by the procedure of (Eisenach, et al.,J. Mol. Biol., 179:125-142 (1986)) with slight modifications. BCG DNAwas digested to completion with BamH I and shotgun cloning of thesefragments into the BamH I site of pBR322 was performed. The genomiclibrary was transformed into E. coli strain DHI and recombinants werescored on the basis of ampicillin resistance and tetracyclinesensitivity. The aim of this approach was to generate restrictionfragments of a broad size range so as not to restrict the library to DNAfragments of any particular size range. This cloning strategy alsoensured to a large extent that any recombinants selected for expressionof mycobacterial antigens should be likely to drive expression from amycobacterial promoter rather than the Tet promoter of pBR322.

[0041] The BCG library constructed in this manner contained 2051 clonesof BCG origin. In an analogous manner, a genomic library ofMycobacterium tuberculosis H37Rv DNA was constructed and 1100 clonesobtained.

[0042] The BCG DNA inserts ranged in size from 0.9 to 9.5 kb. Theaverage size of the mycobacteria DNA fragments inserted in pBR322 wasestimated to be about 4 kb. Given the genome size of BCG to be 4.5×10³kb (Bradley, S. G., J. Bacteriol., 113:645-651 (1973); Imaeda, et al.,Int. J. Syst. Bacteriol., 32, 456-458 (1982)), about 1000 clones of thisaverage insert size would represent comprehensively the entire genome ofthe microorganism.

[0043] B. Isolation of Recombinant DNA Clones Encoding BCG Mycobacteriumbovis BCG and Mycobacterium tuberculosis H37Rv Protein Antigens

[0044] In order to identify recombinants expressing mycobacterialantigens, a colony immunoscreening assay (CIA) to screen recombinantcolonies with appropriate antisera, was established. Sera obtained from20 patients newly diagnosed with active pulmonary tuberculosis werepooled for use in immunoscreening. None of the patients had receivedtreatment for tuberculosis prior to this study and their sputa werepositive for acid fast bacteria in all cases. Pooled sera were absorbedon a E. coli sonicate overnight at 4° C., to eliminate antibodiescross-reactive to E. coli antigens, thereby improving signal to noiseratio during the immunoscreening.

[0045] Individual recombinant colonies were grown overnight onnitrocellulose membranes and immunoscreening was carried out asdescribed with slight modifications. The colonies were lysed inchloroform vapor to release the cloned mycobacterial antigens,immobilized on the nitrocellulose paper. The immobilized antigens werereacted with TB sera and binding of the antibody was revealed bystandard procedures using a horseradish peroxidase-protein A detectionsystem. The signals obtained with the recombinant clones were comparedwith that obtained in case of E. coli colonies harbouring pBR322 vectoralone, which served as the negative control, to assess the signal tonoise ratio. Further, to ascertain whether the immunoreactivity of therecombinant clones was due to anti-mycobacterial antibodies or due to areaction with normal serum components, another CIA of the selectedrecombinants was performed using TB sera and normal human sera NHS whichhad been absorbed on E. coli in a manner analogous to that describedearlier for TB sera. Only those clones reacting selectively with TB seraand not with NHS, were considered to be unambiguously suggestive of thepresence of mycobacterial antigens. The use of this immunoscreeningapproach to identify recombinant colonies carrying mycobacterial DNAinserts capable of expressing mycobacterial antigens is described below:

[0046]FIG. 1 shows the result of immunoscreening of recombinant coloniescarrying M. bovis BCG DNA (panel A) or M. tuberculosis H37 Rv DNA (panelB) using sera from TB patients. The colonies were grown onnitrocellulose paper overnight, lysed to release the clonedmycobacterial antigen and allowed to react with the antibodies. Thepresence of mycobacterial antigen is indicated by a qualitative signalin the recombinant clones which is absent in the negative controlcomprising colonies harbouring pBR322 vector alone. A similar assay wasrepeated with normal human serum to ascertain the specificity of thecloned mycobacterial antigens. 51 recombinant colonies carrying M. bovisBCG DNA inserts and 45 recombinant colonies carrying M. tuberculosisH37Rv DNA inserts were screened by the above procedure; 14 clones of BCGorigin (panel A) and 2 clones of H37Rv origin (panel B) exhibiteddistinct strong signals indicating the immunoreactivity of these clones(FIG. 1). All these clones were also tested for immunoreactivity withNHS. However, with the exception of 3 clones which showed a slightreactivity to NHS, none of the clones reacted with NHS, therebyindicating that these expressed mycobacterial antigens reactedselectively with TB sera. Thus, this procedure resulted in theforthright identification of recombinant clones encoding mycobacterialantigens. This strategy can be generally applicable to mycobacterialgene banks prepared in plasmid or cosmid vectors to identify genes whichare expressed in E. coli at least to the limit detectable by theimmunoassay.

[0047] C. Restriction Mapping of Immunoreactive Mycobacterium bovis BCGDNA Recombinants

[0048] The insert DNAs of four of the immunoreactive BCG recombinant DNAclones isolated using the TB sera were mapped with restrictionendonucleases. FIG. 2, panel B, shows the genomic DNA restriction sitemaps deduced for the cloned BCG DNA in four recombinants, in which, Arepresents Sal I, B, BamH I, E, EcoR I, G, Bgl II, K, Kpn I, P, Pvu I,S, Sac I, X, Xho I. These restriction site maps were then compared withthose constructed previously for the five immunodominant antigens of M.tuberculosis/M. bovis BCG (Young, et al., Proc. Natl. Acad. Sci.,U.S.A., 82:2583-2587 (1985); Husson, et al., Proc. Natl. Acad. Sci.,84:1679-1683 (1987); Shinnick, et al., Infect. Immun., 55, 7:1718-1721(1987)) (FIG. 2, panel A). Since the restriction site maps shown inpanels A and B have been drawn to the same scale, the differencesbetween the two are apparent. There are no regions of similarity betweenthe restriction site maps of immunoreactive BCG recombinant clones andthose of the previously characterized immunodominant antigens of M.tuberculosis/M. bovis BCG. Therefore, one can conclude that the clonedBCG DNA inserts in the four recombinants are novel.

EXAMPLE II Isolation and Characterization of a Gene Encoding a BCGIon-motive ATPase

[0049] A. Identification of a Novel BCG Antigen

[0050] One of the four immunoreactive BCG clones, pMBB51A, revealed thepresence of a protein of Mr 90 kD, on Western blot analysis using TBsera as well as anti-H37Rv polyclonal antiserum raised in rabbits (FIG.3). Similar Western blot analysis of pMBB51A with a pool of a fewanti-mycobacterial monoclonal antibodies (TB 23, TB 71, TB 72, TB 68, TB78; Engers et al., Infec. Immun., 48:603-605 (1985)) or with normalhuman sera did not reveal this immunoreactive protein of 90 kD. Thisconfirms that pMBB51A encodes a BCG antigen which is different fromthose identified previously in BCG, thereby making it a novel antigen.

[0051] B. Determination of the Nucleotide Sequence of pMBB51A

[0052] In order to further characterize this novel BCG antigen, pMBB51ADNA insert was subjected to nucleotide sequencing. The BamH I-BamH Iinsert carried in pMBB51A was mapped for additional restriction enzymecleavage sites. It was determined that there were at a minimum a singlePst I site and 3 Sal I sites in this sequence. Overlapping fragmentsderived from single and double digests of Sal I, BamH I and Sal I, BamHI and Pst I, and Pst I and Sal I, were subcloned into M13mpl8 andM13mpl9 vectors, in preparation for DNA sequence analysis. DNAsequencing was then carried out using commercially available kits suchas the Sequenase system and the T7 system from Pharmacia.Oligonulceotides derived from the determined sequence were synthesizedand used as primers to complete the sequence of the larger inserts.Several areas of compression were encountered during the sequencing andthese were resolved by using dITP in the sequencing reactions, and bychanging the reaction conditions. The complete nucleotide sequence ofthe pMBB51A insert DNA was determined by sequencing both the strandsusing dGTP as well as dITP. The DNA sequence of the pMBB51A insert wasdetermined to be 3.25 kb long with a GC content of 67.1% and is shown inFIG. 4.

[0053] The determination of the DNA sequence of the 3.25 kb insert ofclone pMBB51A (FIG. 4) permitted the elucidation of the amino acidsequence of the 90 kD BCG antigen. In FIG. 4, nucleotides are numberedfrom the left end of the pMBB51A insert DNA.

[0054] A search of pMBB51A insert DNA sequence for possible ORFs in allthree reading frames revealed the longest ORF of 2286 bp encoding apolypeptide of 761 amino acids on one of the strands. The other strandwas found to have a smaller URF of 1047 bp capable of encoding apolypeptide of 349 amino acids. The longest ORF encoding a 761 aminoacid long protein corresponded to a deduced molecular weight of 79 kDwhich came closest to the immunoreactive BCG protein with apparentmolecular weight of 90 kD, seen on the Western blot. The deduced aminoacid sequence for this protein is given below the nucleotide sequence inFIG. 4.

[0055] The location of this ORF on the pMBB51A insert DNA was such thatthere were long stretches of flanking DNA sequences, devoid of anymeaningful ORFs, present on either side. This precluded the expressionof this ORF from the pBR322 Tet gene promoter and instead suggested thatthis ORF was being expressed from its own promoter in pMBB51A. This alsosuggested that E. coli may correctly utilize the M. bovis BCGtranscription and translation start and stop sites in this gene.

[0056] Immediately upstream of the ORF, regulatory sequences closelymatching the −35, −10 and Shine-Dalgarnó sequences of E. coli,(Rosenberg, et al., Annul. Rev. Genet., 13:319-353 (1979)) wereidentified. The spacing between these three regulatory motifs was alsovery well conserved. Although the other mycobacterial promoterssequenced (Dale, et al., Molecular Biology of the Mycobacteria, chap. 8,173-198 (1990)) show some differences from the E. coli consensussequences in all the three regions −35, −10 and SD, the regulatoryelements of pMBB51A DNA showed a maximum degree of sequence identitywith E. coli in the −35 and SD sequence elements with a single mismatchin each element, and about 50% sequence identity in the Pribnow box. Allthe above features clearly indicated that this region is the promoterregion for the mycobacterial gene contained in pMBB51A. The extent ofsimilarity between this BCG promoter sequence and a typical E. colipromoter is remarkable and explains the functional activity of thispromoter, unlike many other mycobacterial promoters, in E. coli. Thetranslation initiation codon in this ORF was ATG at position 508 while asingle translation termination codon TGA was identified at position2790. Potential transcription termination structures capable of formingstem and loop conformations were identified in the region 3′ to thisORF. The pMBB51A ORF thus represented a monocistronic gene rather thanan operon. The promoter region of MBB51A gene is capable of directinggene expression in E. coli as well as in mycobacteria. This promotersequence is useful for directing expression of mycobacterial genes in E.coli. Further, this promoter sequence can also be used to expresshomologous and/or heterologous genes in a mycobacterium, thus providinga key element for the development of gene expression systems inmycobacteria.

[0057] In order to derive information about the possible biologicalfunction of the MBB51A protein, the amino acid sequence of this proteinwas used to search for homology against available sequences in the PIRProtein Database Release 20 (Table I) and a Genebank Nucleic AcidDatabase (Table II) using the Fast A suite of programmes written by(Lipman and Pearson, Proc. Natl. Acad. Sci., USA, 85:2 (1988)). TheMBB51A protein sequence exhibited homology to a family of ion-motiveATPases from different organisms, ranging from bacteria to mammals. The13 best scores from a search with ktuple 2 are shown in the upper panelof Table I and 10 best scores from a search with ktuple 1 are shown inthe lower panel. In each case, MBB51A protein exhibited maximum homology(75.9% homology in a 593 amino acid overlap with 31.9% identity to a K+transporting ATPase of S. faecalis (Solioz et al., 1987). The next besthomology was observed with the B-chain of K+ transporting ATPase of E.coli (Hesse, et al., Proc. Natl. Acad. Sci., U.S.A., 81:4746-4750(1984)) (68.8% homology in a 397 amino acid overlap with 24.2%identity). A lesser extent of homology was also seen with H+, Ca++ andNa+-ATPases from different organisms. The results of homology searchthus indicated that MBB51A protein is an ion-motive ATPase of M. bovisBCG and is closely related to the other bacterial ion-motive ATPases.This is the first report of the cloning and identification of such anATPase in mycobacteria. The BCG ion-motive ATPase showed homologies withother ion-motive ATPases with overlapping regions ranging in size from593 amino acids in case of S. faecalis to 82 amino acids as in case ofL. donovani, (Meade, et al., Mol. Cell Biol., 7, 3937-3946 (1987)),though most of the regions of sequence identity or conservation werelocalized in the C-terminal half of the MBB51A protein. Further, aregion of 30 amino acids in the C-terminal half of MBB51A protein wasfound to be shared with most of these ATPases, thereby suggesting thefunctional importance of this region. Detailed alignment of MBB51Aprotein with the K+ ATPases of S. faecalis and E. coli also indicatedthat several residues were conserved between the three ATPases,including the ones that are invariant in all ATPases from bacteria toman. TABLE I RESULTS OF HOMOLOGY SEARCH OF MBB51A AMINO ACID SEQUENCEAGAINST PIR PROTEIN DATABASE LOCUS SHORT DEFINITION initn opt ktuple :2 >A29576 Potassium - transporting ATPase Strepto- 547 792coccus >PWECBK Potassium - transporting ATPase, β chain - 314 270 E.coli >A25939 Proton - transporting ATPase - Neurospora 168 186 >A25823Proton - transporting ATPase - Yeast 166 184 >PWRBFC Calcium -transporting ATPase, fast twitch 152 158 skele >PWRBSC Calcium -transporting ATPase, slow twitch 135 157 skele >A25344 Potassium -transporting ATPase - Rat 78 155 >RDEBHA Mercuric reductase -Shigellaflexneri 99 142 plasmid >RDPSHA Mercuric reductase (transposon Tn501) 74124 >RGPSHA Mercuric resistance operon regulatory p 79 109 >A24639Sodium/potassium-transporting ATPase, 92 82 alpha >A24414Sodium/potassium-transporting ATPase, 92 82 alpha >B24862Sodium/potassium-transporting ATPase, beta 83 82

[0058] The PJR protein data base (2378611 residues in 9124 sequences)was scanned with the FASTA program. The mean of the original initialscore was 27.2 with a standard deviation of 6.9. Initial scores (initn)higher than 75.6 are 6 standard deviations above the average, a level ofsignificance that usually indicates biological relatedness. Optimization(opt) generally will improve the initial score of related proteins byintroducing gaps in the sequence. Unrelated sequences usually do nothave their scores improved by optimization. ktuple : 1 >A29576potassium-transporting ATPase - Strepto- 744 792 coccus >PWECBKpotassium-transporting ATPase, β chain - 386 270 Esche >A25939 Proton-transporting ATPase - Neurospora 310 186 crassa >A25823proton-transporting ATPase -Yeast (Sacchar- 317 184 omy) >B24639Sodium/potassium-transporting ATPase, al- 158 163 pha (+ >A24639Sodium/potassium-transporting ATPase, al- 175 160 pha ch >C24639Sodium/potassium-transporting ATPase, al- 192 159 pha (II >PWRBFCCalcium-transporting ATPase, fast twitch 240 158 skele >PWSHNASodium/potassium-transporting ATPase, al- 214 158 pha skele >A24414Sodium/potassium-transporting ATPase, al- 214 158 pha chain

[0059] TABLE II RESULTS OF HOMOLOGY SEARCH OF MBB51A AMINO ACID SEQUENCEAGAINST GENBANK NUCLEIC ACID SEQUENCE DATABASE LOCUS SHORT DEFINITIONinitn opt ktuple : 2 >STRATPK S. faecalis K+ ATPase, complete cds. 537800 >ECOKDPABC E. coli kdpABC operon coding for Kdp- 314 270ATpase >YSPPMA1A S. pombe H+ ATPase, complete cds. 135 188 >NEUATPASE N.crassa plasma membrane ATPase, 133 186 complete >NEUATPPM Neurosporacrassa plasma membrane 131 186 H+ ATPase >YSCPMA1 Yeast PMA1 for plasmamembrane 166 184 ATPase >M17889 FIG. 2 N of L. donovani ATPase and 166170 >M12898 Rabbit fast twitch skeletal muscle Ca++ 140 158ATPas >RABATPAC Rabbit Ca + Mg dependent Ca++ 142 157 ATPase mRNA,co >NR1MER Plasmid NR1 mercury resistance (mer) 100 143 operon. ktuple :1 >STRATPK S. faecalis K+ ATPase gene, 744 800 complete cds. >SYNCATPSBCyanobacterium Synechococcus 6301 379 422 DNA for AT >ECOKDPABC E. colikdpABC operon coding for Kdp- 379 270 ATPase p >YSPPMA1A S. pombe H+ATPase gene, com- 275 188 plete cds. >NEUATPASE N. crassa plasmamembrane ATPase 311 186 gene, comple >NEUATPPM Neurospora crassa plasmamembrane 302 186 H+ ATPase >YSCPMA1 Yeast PMA1 gene for plasma mem- 317184 brane ATPase >JO4004 Leishmania donovani. cation trans- 322 170porting ATP >M17889 FIG. 2 Nucleotide seguence of L. 306 170donovani >RATAPA2 Rat Na+, K+ ATPase alpha (+) isoform 158 163 catalytic

[0060] The KdpB protein of E. coli and possibly the S. faecalis K+ATPase are members of E1E2-ATPases which are known to form an aspartylphosphate intermediate, with cyclic transformation of the enzyme betweenphosphorylated and dephosphorylated species. By analogy to otherATPases, the phosphorylated Asp residue (D) (Furst, et al., J. Biol.Chem., 260:50-52 (1985)) was identified at position 443 in the MBB51AATPase. This residue is the first of a pentapeptide sequence DKTGT thathas been conserved in ATPases from bacteria to man, and must form anessential element of the catalytic site. Similarly, proline (P) atposition 400 in MBB51A ATPase was found to be an invariant amino acid inother ATPases and is predicted to be located in a membrane spanningdomain. Such membrane buried proline residues have been hypothesized tobe required for the reversible conformational changes necessary for theregulation of a transport channel (Brandl, et al., Proc. Natl. Acad.Sci., U.S.A., 83:917-921 (1986)). In addition, other sequence motifsbelieved to be functionally important in other ion-motive ATPases werealso found to be conserved in the MBB51A ATPase. These include a Gly (G)(Farley and Faller, J. Biol. Chem., 260:3899-3901 (1985)) at position521 and Ala (A) (Ohta, et al., Proc. Natl. Acad. Sci., U.S.A.,83:2071-2075 (1986)) at position 646, and are shown in FIG. 5.

[0061] Since the MBB51A ATPase was homologous to membrane associatedATPases, characterization of the membrane associated helices in MBB51Aprotein was performed by computer algorithms. Using a hydropathy profile(Rao, et al., Biochem. Biophys. Acta., 869:197-214 (1986)), seventransmembrane domains in the MBB51A protein were identified and areshown in Table III and FIG. 5. Nearly the same transmembrane domainswere also identified using the hydrophobic moment plot (Eisenberg etal., J. Mol. Biol., 179:125-142 (1984)) and are also shown in Table IIIand FIG. 5. The average size of a transmembrane domain is around 21residues, because 21 residues coil into an α-helix approximately thethickness of the apolar position of a lipid bilayer (32 Å). This size ofa transmembrane domain is, however, flexible within the range of a fewamino acids, as determined by the functional properties of a givenmembrane-associated protein. The transmembrane domains identified inMBB51A protein, range in size from 20-37 residues. The first sixtransmembrane domains span the membrane only once, as indicated by boththe hydropathy profile and the hydrophobic moment plot. The seventhtransmembrane domain may traverse the membrane twice. These featuresalong with the membrane buried proline (P) at position 400, are inaccordance with the channel transport functions of ion-motive ATPases,involving a reversible change in the conformation of these proteins.Such transmembrane domains further define the intracellular andextracellular domains of this molecule. See FIG. 5. TABLE IIITransmembrane Eisenberg Rao & Argos Domain in FIG. 5 Method Method 1102-122  98-125 2 129-149 127-147 3 164-184 164-185 4 199-219 198-220 5361-381 360-382 6 387-407 387-419 7 703-723 695-732

[0062] The hydropathy profile of MBB51A protein was nearlysuperimposable over that of S. faecalis K+ ATPase, even though theMBB51A ATPase has at the N-terminus, 154 extra amino acids, which wereabsent in S. faecalis. This clearly puts in evidence the strongevolutionary conservation of the broad domain structure between thesetwo proteins, making it more likely for the two proteins to have asimilar three dimensional structural organization.

[0063] Based on the hydropathy profile and secondary structurepredictions, a schematic model of the MBB51A ATPase is presented in FIG.5. This model comprises at least seven transmembrane domains which spanthe membrane, once are indicated along with the respective amino acidpositions in FIG. 5. This model further defines extracellular andintracellular domains of the MBB51A protein. Many of the residues whichhave been shown to be functionally important in other ion-motive ATPasesand are also conserved in the MBB51A protein, are also shown. Of these,proline (P) at position 400 is membrane-buried whereas as asparticacid(D) at 443, glycine (G) at 521 and alanine (A) at 646, face thecytoplasm.

[0064] In order to determine whether the gene encoding MBB51A ion-motiveATPase is present in other mycobacterial strains related or unrelated toBCG, like the virulent strain M. tuberculosis H37Rv and othernon-tuberculous, non-pathogenic mycobacteria like M. vaccae and M.smegmatis, Southern blot hybridization with genomic DNA from the abovespecies was performed, using as probe BCG insert DNA from pMBB51A. Asshown in FIG. 6, DNA hybridizable with the pMBB51A insert DNA was alsopresent in M. tuberculosis H37Rv DNA but not in M. smegmatis and M.vaccae. This indicated that the M. tuberculosis H37Rv homologue of thepMBB51A gene has a similar genetic organization as seen in M. bovis BCGDNA, and is present on a 3.25 kb BamH I fragment.

[0065] The availability of novel Mycobacterium bovis BCG and/orMycobacterium tuberculosis H37Rv antigens make it possible to addressbasic biochemical, immunological, diagnostic and therapeutic questionsstill unanswered about tuberculosis and Mycobacterium tuberculosis. Forexample, Mycobacterium tuberculosis specific antigenic determinants canbe used to develop simple and specific seroepidemiological tests toscreen human populations. Such serological tests are highly specificbecause of the use of antigenic determinants determined by theapproaches described above and known to be unique to Mycobacteriumtuberculosis H37Rv. Such serological tests are useful for earlydiagnosis of tuberculosis, thus permitting early treatment and limitingtransmission of the disease from infected individuals to others.

[0066] Resistance to tuberculosis is provided by cell mediated immunity.The antigens identified here can be further used to determine whichsegments of these antigens are recognized by Mycobacterium tuberculosisspecific T-cells. A mixture of peptides recognized by helper T-cellsprovides a specific skin test antigen for use in assessing theimmunological status of patients and their contacts. A mixture of suchpeptides is also useful in evaluating rapidly the immunological efficacyof candidate vaccines. In addition peptides recognized by Mycobacteriumtuberculosis specific T-cells can be components of a vaccine against thedisease.

[0067] Knowledge of the complete nucleotide sequence of pMBB51A DNAinsert provides a rich source of sequence information which can be usedto design appropriate primers for PCR amplification of mycobacterialgenomic DNA fragments. The ion-motive ATPase of BCG has areas of heavilyconserved sequences (for, e.g., the ATP binding site) which are expectedto be the same for all mycobacterial species and areas of sequencedivergence (for, e.g., the N-terminal region) which are different indifferent mycobacterial species. Based on this knowledge primers can bedesigned either from the conserved regions or from the diverged regionsto identify whether in a given sample the target DNA is mycobacterialversus non-mycobacterial, and in case of mycobacterial DNA, whichmycobacterial species the DNA belongs.

[0068] Such amplification schemes are useful for the development ofhighly sensitive and specific PCR amplification based diagnosticprocedures for mycobacteria. The observation that the 3.25 kb pMBB51ADNA insert is present in Mycobacterium tuberculosis H37Rv andMycobacterium bovis BCG and is absent in avirulent Mycobacterium vaccaeand Mycobacterium smegmatis, which have bearing on other aspects of thebiological differences between these species, manifest in terms ofvirulence, growth characteristics and metabolism.

[0069] Recombinant vaccines can also be constructed by incorporating theDNA encoding all or part of the membrane-associated polypeptides of theinvention into an appropriate vaccine vehicle. For example, all or partof the DNA encoding the 79 kD Mycobacterium bovis BCG protein or aportion of the protein can be incorporated into a vaccine vehiclecapable of expressing the said DNA. Such a vaccine vehicle could be avirus for, e.g., vaccinia virus, etc., or a bacterium, e.g.,mycobacteria, Salmonella, Vibrio, Bacillus, Yersinia, Bordetella, etc.to produce a vaccine capable of conferring long-lasting immunity onindividuals to whom it is administered.

[0070] A special feature of the 79 kD BCG ion-motive ATPase is that itis a membrane bound antigen. Therefore, it can be used to link foreignDNA sequences encoding antigenic epitopes (B-cell epitopes or T-cellepitopes) of interest, with this gene or a portion of this gene in amanner which causes the foreign epitope to be used as an immunogen. Suchlinkages can be engineered into extracellular or intracellular domainsof MBB51A protein, or into a combination of both types of domains.Engineering of immunogenic foreign epitopes into MBB51A DNA isaccomplished by standard recombinant DNA methods known to those skilledin the art. Some of these methods involve use of unique restrictionsites, in vitro mutagenesis and/or PCR-related methods. One suchconvenient method involves the use of a unique NdeI site at position1090 in the MBB51A DNA where foreign DNA can be inserted. Grafting ofepitopes on the cell surface induces rapid antibody response by virtueof the epitope being well-exposed on the bacterial cell, which in turnleads to direct activation of B cells. In addition, intracellularlocalization of an epitope induces B cell memory and a proficient T cellresponse. Examples of epitopes of interest known to be involved in theimmune response to various pathogens include epitopes from E. coli LTtoxin, foot and mouth disease virus, HIV, cholera toxin, etc.

[0071] Thus, the 79 kD antigen is useful in the design of recombinantvaccines against different pathogens. Such vaccines comprise arecombinant vaccine vehicle capable of expressing all or part of the 79kD membrane-associated protein of mycobacteria, into which foreignepitopes have been engineered, such that the foreign epitopes areexpressed on the outer surface and/or on the inner side of the cellmembrane, thereby rendering the foreign epitopes immunogenic. Thevaccine vehicle for this purpose may be a cultivable mycobacterium for,e.g., BCG. In these applications, the BCG ion-motive ATPase gene can beborne on a mycobacterial shuttle vector or alternately the foreign DNAencoding antigenic epitopes of the immunogenic polypeptides can beinserted into the mycobacterial genome via homologous recombination inthe ion-motive ATPase gene or random integration. Such a process yieldsstable recombinant mycobacterial strains capable of expressing on theirsurface and/or in the cytoplasm antigenic sequences of interest, whichcan, for example, provide protection against a variety of infectiouspathogens. Targeting of recombinant antigens to the cell-wall isattractive not only because of the high immunogenicity of mycobacterialcell-walls but, in addition, because of concerns with the introductionof a live vaccine in populations with a high prevalence of HIVseropositivity. Additionally, based on the MBB51A protein, a non-livingbut immunogenic recombinant cell surface subunit vaccine can also bedeveloped to provide a useful alternative to live vaccines. Alternately,other bacterial, viral or protozoan vaccine vehicles could betransformed to generate such recombinant vaccines. Examples of potentialvaccine vehicles include vaccinia virus, pox-viruses, Salmonella,Yerisinia, Vibrio, Bordetella, Bacillus, etc.

[0072] Further, using such an approach, multivalent recombinant vaccineswhich allow simultaneous expression of multiple protectiveepitopes/antigens of different pathogens, could also be designed.

[0073] Equivalents

[0074] Those skilled in the art will recognize, or be able to ascertain,using no more than routine experimentation, many equivalents to thespecific materials and components described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

1 2 1 3250 DNA Mycobacterium bovis CDS (508)..(2790) 1 gatcccgcggtcatcgatc gggtcaaaca ccgcctcgac gggttcacgc tggcgccgct 60 gtccaccgccgcgggaggtg gtggccggca gccacgcatc tactacggca ccatcctgac 120 cggtgaccaataccttcact gcgagcgcac ccgcaaccgg ctgcaccacg aactcggcgg 180 tatggccgtcgaaatggaag gcggtgcggt ggcgcaaatc tgcgcgtcct tcgatatccc 240 atggctggtcattcgcgcgc tctccgatct cgccggagcc gattcggggg tggacttcaa 300 tcggtttgtcggcgaggtgg cggccagttc ggcccgcgtt ctgctgcgct tgctgccggt 360 gttgacggcctgttgaagac gactatccgc cggtgcgttc accgcgtcag gcggcttcgg 420 tgaggtgagtaatttggtca ttaacttggt catgccgccg ccgatgttga gcggaggcca 480 caggtcggccggaagtgagg agccacg atg acg gcg gcc gtg acc ggt gaa cac 534 Met Thr AlaAla Val Thr Gly Glu His 1 5 cac gcg agt gtg cag cgg ata caa ctc aga atcagc ggg atg tcg tgc 582 His Ala Ser Val Gln Arg Ile Gln Leu Arg Ile SerGly Met Ser Cys 10 15 20 25 tct gcg tgc gcc cac cgt gtg gaa tcg acc ctcaac aag ctg ccg ggg 630 Ser Ala Cys Ala His Arg Val Glu Ser Thr Leu AsnLys Leu Pro Gly 30 35 40 gtt cgg gca gct gtg aac ttc ggc acc cgg gtg gcaacc atc gac acc 678 Val Arg Ala Ala Val Asn Phe Gly Thr Arg Val Ala ThrIle Asp Thr 45 50 55 agc gag gcg gtc gac gct gcc gcg ctg tgc cag gcg gtccgc cgc gcg 726 Ser Glu Ala Val Asp Ala Ala Ala Leu Cys Gln Ala Val ArgArg Ala 60 65 70 ggc tat cag gcc gat ctg tgc acg gat gac ggt cgg agc gcgagt gat 774 Gly Tyr Gln Ala Asp Leu Cys Thr Asp Asp Gly Arg Ser Ala SerAsp 75 80 85 ccg gac gcc gac cac gct cga cag ctg ctg atc cgg cta gcg atcgcc 822 Pro Asp Ala Asp His Ala Arg Gln Leu Leu Ile Arg Leu Ala Ile Ala90 95 100 105 gcc gtg ctg ttt gtg ccc gtg gcc gat ctg tcg gtg atg tttggg gtc 870 Ala Val Leu Phe Val Pro Val Ala Asp Leu Ser Val Met Phe GlyVal 110 115 120 gtg cct gcc acg cgc ttc acc ggc tgg cag tgg gtg cta agcgcg ctg 918 Val Pro Ala Thr Arg Phe Thr Gly Trp Gln Trp Val Leu Ser AlaLeu 125 130 135 gca ctg ccg gtc gtg acc tgg gcg gcg tgg ccg ttt cac cgcgtt gcg 966 Ala Leu Pro Val Val Thr Trp Ala Ala Trp Pro Phe His Arg ValAla 140 145 150 atg cgc aac gcc cgc cac cac gcc gcc tcc atg gag acg ctaatc tcg 1014 Met Arg Asn Ala Arg His His Ala Ala Ser Met Glu Thr Leu IleSer 155 160 165 gtc ggt atc acg gcc gcc acg atc tgg tcg ctg tac acc gtcttc ggc 1062 Val Gly Ile Thr Ala Ala Thr Ile Trp Ser Leu Tyr Thr Val PheGly 170 175 180 185 aat cac tcg ccc atc gag cgc agc ggc ata tgg cag gcgctg ctg gga 1110 Asn His Ser Pro Ile Glu Arg Ser Gly Ile Trp Gln Ala LeuLeu Gly 190 195 200 agc gat gct att tat ttc gag gtc gcg gcg ggt gtc acggtg ttc gtg 1158 Ser Asp Ala Ile Tyr Phe Glu Val Ala Ala Gly Val Thr ValPhe Val 205 210 215 ctg gtg ggg cgg tat ttc gag gcg cgc gcc aag tcg caggcg ggc agt 1206 Leu Val Gly Arg Tyr Phe Glu Ala Arg Ala Lys Ser Gln AlaGly Ser 220 225 230 gcg ctg aga gcc ttg gcg gcg ctg agc gcc aag gaa gtagcc gtc ctg 1254 Ala Leu Arg Ala Leu Ala Ala Leu Ser Ala Lys Glu Val AlaVal Leu 235 240 245 cta ccg gat ggg tcg gag atg gtc atc ccg gcc gac gaactc aaa gaa 1302 Leu Pro Asp Gly Ser Glu Met Val Ile Pro Ala Asp Glu LeuLys Glu 250 255 260 265 cag cag cgc ttc gtg gtg cgt cca ggg cag ata gttgcc gcc gac ggc 1350 Gln Gln Arg Phe Val Val Arg Pro Gly Gln Ile Val AlaAla Asp Gly 270 275 280 ctc gcc gtc gac ggg tcc gct gcg gtc gac atg agcgcg atg acc ggc 1398 Leu Ala Val Asp Gly Ser Ala Ala Val Asp Met Ser AlaMet Thr Gly 285 290 295 gag gcc aaa ccg acc cgg gtg cgt ccg ggg ggg caggtc atc ggc ggc 1446 Glu Ala Lys Pro Thr Arg Val Arg Pro Gly Gly Gln ValIle Gly Gly 300 305 310 acc aca gtg ctt gac ggc cgg ctg atc gtg gag gcggcc gcg gtg ggc 1494 Thr Thr Val Leu Asp Gly Arg Leu Ile Val Glu Ala AlaAla Val Gly 315 320 325 gcc gac acc cag ttc gcc gga atg gtc cgc ctc gttgag caa gcg cag 1542 Ala Asp Thr Gln Phe Ala Gly Met Val Arg Leu Val GluGln Ala Gln 330 335 340 345 gcg caa aag gcc gac gca cag cga cta gcc gaccgg atc tcc tcg gtg 1590 Ala Gln Lys Ala Asp Ala Gln Arg Leu Ala Asp ArgIle Ser Ser Val 350 355 360 ttt gtt ccc gct gtg ttg gtt atc gcg gca ctaacc gca gcc gga tgg 1638 Phe Val Pro Ala Val Leu Val Ile Ala Ala Leu ThrAla Ala Gly Trp 365 370 375 cta atc gcc ggg gga caa ccc gac cgt gcc gtctcg gcc gca ctc gcc 1686 Leu Ile Ala Gly Gly Gln Pro Asp Arg Ala Val SerAla Ala Leu Ala 380 385 390 gtg ctt gtc atc gcc tgc ccg tgt gcc ctg gggctg gcg act ccg acc 1734 Val Leu Val Ile Ala Cys Pro Cys Ala Leu Gly LeuAla Thr Pro Thr 395 400 405 gcg atg atg gtg gcc tct ggt cgc ggt gcc cagctc gga ata ttt ctg 1782 Ala Met Met Val Ala Ser Gly Arg Gly Ala Gln LeuGly Ile Phe Leu 410 415 420 425 aag ggc tac aaa tcg ttg gag gcc acc cgcgcg gtg gac acc gtc gtc 1830 Lys Gly Tyr Lys Ser Leu Glu Ala Thr Arg AlaVal Asp Thr Val Val 430 435 440 ttc gac aag acc ggc acc ctg acg acg ggccgg ctg cag gtc agt gcg 1878 Phe Asp Lys Thr Gly Thr Leu Thr Thr Gly ArgLeu Gln Val Ser Ala 445 450 455 gtg acc gcg gca ccg ggc tgg gag gcc gaccag gtg ctc gcc ttg gcc 1926 Val Thr Ala Ala Pro Gly Trp Glu Ala Asp GlnVal Leu Ala Leu Ala 460 465 470 gcg acc gtg gaa gcc gcg tcc gag cac tcggtg gcg ctc gcg atc gcc 1974 Ala Thr Val Glu Ala Ala Ser Glu His Ser ValAla Leu Ala Ile Ala 475 480 485 gcg gca acg act cgg cga gac gcg gtc accgac ttt cgc gcc ata ccc 2022 Ala Ala Thr Thr Arg Arg Asp Ala Val Thr AspPhe Arg Ala Ile Pro 490 495 500 505 ggc cgc ggc gtc agc ggc acc gtg tccggg cgg gcg gta cgg gtg ggc 2070 Gly Arg Gly Val Ser Gly Thr Val Ser GlyArg Ala Val Arg Val Gly 510 515 520 aaa ccg tca tgg atc ggg tcc tcg tcgtgc cac ccc aac atg cgc gcg 2118 Lys Pro Ser Trp Ile Gly Ser Ser Ser CysHis Pro Asn Met Arg Ala 525 530 535 gcc cgg cgc cac gcc gaa tcg ctg ggtgag acg gcc gta ttc gtc gag 2166 Ala Arg Arg His Ala Glu Ser Leu Gly GluThr Ala Val Phe Val Glu 540 545 550 gtc gac ggc gaa cca tgc ggg gtc atcgcg gtc gcc gac gcc gtc aag 2214 Val Asp Gly Glu Pro Cys Gly Val Ile AlaVal Ala Asp Ala Val Lys 555 560 565 gac tcg gcg cga gac gcc gtg gcc gccctg gcc gat cgt ggt ctg cgc 2262 Asp Ser Ala Arg Asp Ala Val Ala Ala LeuAla Asp Arg Gly Leu Arg 570 575 580 585 acc atg ctg ttg acc ggt gac aatccc gaa tcg gcg gcg gcc gtg gct 2310 Thr Met Leu Leu Thr Gly Asp Asn ProGlu Ser Ala Ala Ala Val Ala 590 595 600 act cgc gtc ggc atc gac gag gtgatc gcc gac atc ctg ccg gaa ggc 2358 Thr Arg Val Gly Ile Asp Glu Val IleAla Asp Ile Leu Pro Glu Gly 605 610 615 aag gtc gat gtc atc gag cag ctacgc gac cgc gga cat gtc gtc gcc 2406 Lys Val Asp Val Ile Glu Gln Leu ArgAsp Arg Gly His Val Val Ala 620 625 630 atg gtc ggt gac ggc atc aac gacgga ccc gca ctg gcc cgt gcc gat 2454 Met Val Gly Asp Gly Ile Asn Asp GlyPro Ala Leu Ala Arg Ala Asp 635 640 645 cta ggc atg gcc atc ggg cgc ggcacg gac gtc gcg atc ggt gcc gcc 2502 Leu Gly Met Ala Ile Gly Arg Gly ThrAsp Val Ala Ile Gly Ala Ala 650 655 660 665 gac atc atc ttg gtc cgc gaccac ctc gac gtt gta ccc ctt gcg ctt 2550 Asp Ile Ile Leu Val Arg Asp HisLeu Asp Val Val Pro Leu Ala Leu 670 675 680 gac ctg gca agg gcc acg atgcgc acc gtc aaa ctc aac atg gtc tgg 2598 Asp Leu Ala Arg Ala Thr Met ArgThr Val Lys Leu Asn Met Val Trp 685 690 695 gca ttc gga tac aac atc gccgcg att ccc gtc gcc gct gcc gga ctg 2646 Ala Phe Gly Tyr Asn Ile Ala AlaIle Pro Val Ala Ala Ala Gly Leu 700 705 710 ctc aac ccc ctg gtg gcc ggtgcg gcc atg gcg ttc tca tcg ttc ttc 2694 Leu Asn Pro Leu Val Ala Gly AlaAla Met Ala Phe Ser Ser Phe Phe 715 720 725 gtg gtc tca aac agc ttg cggttg cgc aaa ttt ggg cga tac ccg cta 2742 Val Val Ser Asn Ser Leu Arg LeuArg Lys Phe Gly Arg Tyr Pro Leu 730 735 740 745 ggc tgc gga acc gtc ggtggg cca caa atg acc gcg ccg tcg tcc gcg 2790 Gly Cys Gly Thr Val Gly GlyPro Gln Met Thr Ala Pro Ser Ser Ala 750 755 760 tgatgcgttg tcgggcaacacgatatcggg ctcagcggcg accgcatccg gtctcggccg 2850 aggaccagag gcgcttcgccacaccatgat tgccaggacc gcgccgatca ccaccggcag 2910 atgagtcaaa atccgcgtggtgctgaccgc gccggacagc gcatccacaa tcacatagcc 2970 ggtcagtatg gcgacgaacgccgtcagaac accggccagg ccggcggcgg cgctcggcca 3030 tagcgccgcg cccaccatgatcacaccgag cgcaatcgac cacgacgtga ctcgttgagc 3090 aagtgggtgc cggcacccgtcgggtgctga tgggtcaggc cgacgtctag gccaaacccc 3150 tgcacggtgc ccagggcgatctgcgcgatg cccacgcaca gcaacgccca acgtcgccag 3210 gtcatcggtg aatgttgccgccgcggcgcc cggcggatcc 3250 2 761 PRT Mycobacterium bovis 2 Met Thr AlaAla Val Thr Gly Glu His His Ala Ser Val Gln Arg Ile 1 5 10 15 Gln LeuArg Ile Ser Gly Met Ser Cys Ser Ala Cys Ala His Arg Val 20 25 30 Glu SerThr Leu Asn Lys Leu Pro Gly Val Arg Ala Ala Val Asn Phe 35 40 45 Gly ThrArg Val Ala Thr Ile Asp Thr Ser Glu Ala Val Asp Ala Ala 50 55 60 Ala LeuCys Gln Ala Val Arg Arg Ala Gly Tyr Gln Ala Asp Leu Cys 65 70 75 80 ThrAsp Asp Gly Arg Ser Ala Ser Asp Pro Asp Ala Asp His Ala Arg 85 90 95 GlnLeu Leu Ile Arg Leu Ala Ile Ala Ala Val Leu Phe Val Pro Val 100 105 110Ala Asp Leu Ser Val Met Phe Gly Val Val Pro Ala Thr Arg Phe Thr 115 120125 Gly Trp Gln Trp Val Leu Ser Ala Leu Ala Leu Pro Val Val Thr Trp 130135 140 Ala Ala Trp Pro Phe His Arg Val Ala Met Arg Asn Ala Arg His His145 150 155 160 Ala Ala Ser Met Glu Thr Leu Ile Ser Val Gly Ile Thr AlaAla Thr 165 170 175 Ile Trp Ser Leu Tyr Thr Val Phe Gly Asn His Ser ProIle Glu Arg 180 185 190 Ser Gly Ile Trp Gln Ala Leu Leu Gly Ser Asp AlaIle Tyr Phe Glu 195 200 205 Val Ala Ala Gly Val Thr Val Phe Val Leu ValGly Arg Tyr Phe Glu 210 215 220 Ala Arg Ala Lys Ser Gln Ala Gly Ser AlaLeu Arg Ala Leu Ala Ala 225 230 235 240 Leu Ser Ala Lys Glu Val Ala ValLeu Leu Pro Asp Gly Ser Glu Met 245 250 255 Val Ile Pro Ala Asp Glu LeuLys Glu Gln Gln Arg Phe Val Val Arg 260 265 270 Pro Gly Gln Ile Val AlaAla Asp Gly Leu Ala Val Asp Gly Ser Ala 275 280 285 Ala Val Asp Met SerAla Met Thr Gly Glu Ala Lys Pro Thr Arg Val 290 295 300 Arg Pro Gly GlyGln Val Ile Gly Gly Thr Thr Val Leu Asp Gly Arg 305 310 315 320 Leu IleVal Glu Ala Ala Ala Val Gly Ala Asp Thr Gln Phe Ala Gly 325 330 335 MetVal Arg Leu Val Glu Gln Ala Gln Ala Gln Lys Ala Asp Ala Gln 340 345 350Arg Leu Ala Asp Arg Ile Ser Ser Val Phe Val Pro Ala Val Leu Val 355 360365 Ile Ala Ala Leu Thr Ala Ala Gly Trp Leu Ile Ala Gly Gly Gln Pro 370375 380 Asp Arg Ala Val Ser Ala Ala Leu Ala Val Leu Val Ile Ala Cys Pro385 390 395 400 Cys Ala Leu Gly Leu Ala Thr Pro Thr Ala Met Met Val AlaSer Gly 405 410 415 Arg Gly Ala Gln Leu Gly Ile Phe Leu Lys Gly Tyr LysSer Leu Glu 420 425 430 Ala Thr Arg Ala Val Asp Thr Val Val Phe Asp LysThr Gly Thr Leu 435 440 445 Thr Thr Gly Arg Leu Gln Val Ser Ala Val ThrAla Ala Pro Gly Trp 450 455 460 Glu Ala Asp Gln Val Leu Ala Leu Ala AlaThr Val Glu Ala Ala Ser 465 470 475 480 Glu His Ser Val Ala Leu Ala IleAla Ala Ala Thr Thr Arg Arg Asp 485 490 495 Ala Val Thr Asp Phe Arg AlaIle Pro Gly Arg Gly Val Ser Gly Thr 500 505 510 Val Ser Gly Arg Ala ValArg Val Gly Lys Pro Ser Trp Ile Gly Ser 515 520 525 Ser Ser Cys His ProAsn Met Arg Ala Ala Arg Arg His Ala Glu Ser 530 535 540 Leu Gly Glu ThrAla Val Phe Val Glu Val Asp Gly Glu Pro Cys Gly 545 550 555 560 Val IleAla Val Ala Asp Ala Val Lys Asp Ser Ala Arg Asp Ala Val 565 570 575 AlaAla Leu Ala Asp Arg Gly Leu Arg Thr Met Leu Leu Thr Gly Asp 580 585 590Asn Pro Glu Ser Ala Ala Ala Val Ala Thr Arg Val Gly Ile Asp Glu 595 600605 Val Ile Ala Asp Ile Leu Pro Glu Gly Lys Val Asp Val Ile Glu Gln 610615 620 Leu Arg Asp Arg Gly His Val Val Ala Met Val Gly Asp Gly Ile Asn625 630 635 640 Asp Gly Pro Ala Leu Ala Arg Ala Asp Leu Gly Met Ala IleGly Arg 645 650 655 Gly Thr Asp Val Ala Ile Gly Ala Ala Asp Ile Ile LeuVal Arg Asp 660 665 670 His Leu Asp Val Val Pro Leu Ala Leu Asp Leu AlaArg Ala Thr Met 675 680 685 Arg Thr Val Lys Leu Asn Met Val Trp Ala PheGly Tyr Asn Ile Ala 690 695 700 Ala Ile Pro Val Ala Ala Ala Gly Leu LeuAsn Pro Leu Val Ala Gly 705 710 715 720 Ala Ala Met Ala Phe Ser Ser PhePhe Val Val Ser Asn Ser Leu Arg 725 730 735 Leu Arg Lys Phe Gly Arg TyrPro Leu Gly Cys Gly Thr Val Gly Gly 740 745 750 Pro Gln Met Thr Ala ProSer Ser Ala 755 760

What is claimed is:
 1. Composition comprising recombinant nucleic acidencoding all or part of a membrane-associated polypeptide of amycobacterium, wherein said mycobacterium is capable of inducing animmune response that is detectable with all or part of saidmembrane-associated polypeptide.
 2. The composition of claim 1 whereinsaid mycobacterium is selected from the group consisting of M. bovis, M.tuberculosis, M. leprae, M. africanum, and M. microti, M. avium, M.intracellular and M. scrofulaceum.
 3. The composition of claim 1 whereinsaid mycobacterium is M. bovis BCG.
 4. The composition of claim 3wherein said membrane-associated polypeptide comprises an ion-motiveATPase.
 5. The composition of claim 4 wherein said ATPase has a deducedmolecular weight of about 79 kD.
 6. The composition of claim 1 whereinsaid membrane-associated polypeptide is encoded by a DNA sequencecapable of hybridizing with nucleic acid containing all or part of theDNA SEQUENCE ID NO:
 1. 7. The composition of claim 6 wherein saidnucleic acid encodes at least an extracellular domain of saidmembrane-associated polypeptide.
 8. The composition of claim 6 whereinsaid nucleic acid encodes at least an intracellular domain of saidmembrane-associated polypeptide.
 9. The composition of claim 6 whereinsaid nucleic acid encodes at least one transmembrane domain of saidmembrane-associated polypeptide.
 10. The composition of claim 9 whereinsaid nucleic acid encodes a chimeric polypeptide comprising said atleast one transmembrane domain and an immunogenic polypeptide. 11.Composition comprising all or part of a membrane-associated polypeptideof a mycobacterium, wherein said mycobacterium is capable of inducing animmune response that is detectable with all or part of saidmembrane-associated polypeptide.
 12. The composition of claim 11 whereinsaid mycobacterium is selected from the group consisting of M. bovis, M.tuberculosis, M. leprae, M. africanum, and M. microti, M. arium, M.intracellular and M. scrofulaceum.
 13. The composition of claim 11wherein said mycobacterium is M. bovis BCG.
 14. The composition of claim13 wherein said membrane-associated polypeptide comprises an ion-motiveATPase.
 15. The composition of claim 14 wherein said ATPase has adeduced molecular weight of about 79 kD.
 16. The composition of claim 11wherein said membrane-associated polypeptide is encoded by a nucleicacid capable of hybridizing with a nucleic acid encoding all or part ofDNA SEQUENCE ID NO:1.
 17. The composition of claim 16 wherein saidpolypeptide comprises at least an extracellular domain of saidmembrane-associated polypeptide.
 18. The composition of claim 16 whereinsaid polypeptide comprises at least an intracellular domain of saidmembrane-associated polypeptide.
 19. The composition of claim 16 whereinsaid polypeptide comprises at least one transmembrane domain of saidmembrane-associated polypeptide.
 20. The composition of claim 19 whereinsaid polypeptide comprises a chimeric polypeptide comprising said atleast one transmembrane domain and an immunogenic polypeptide.
 21. Avaccine comprising all or part of a membrane-associated polypeptide of amycobacterium or expressible nucleic acid encoding all or part of saidpolypeptide, in a recombinant vaccine vehicle capable of expressing saidDNA, wherein the vaccine vehicle comprises a virus or a bacterium. 22.The vaccine of claim 21 wherein said membrane-associated polypeptide isan ion-motive ATPase of a mycobacterium.
 23. Nucleic acid comprising apromoter sequence from an ion-motive ATPase of a mycobacterium.